Rep interaction with cellular proteins. Cells which over-express the larger Rep polypeptides have a distinctive phenotype. These cells appear to be blocked in the cell cycle and are unable to undergo mitosis and undergo apoptotic cell death. Recently we have demonstrated that the unspliced forms of the Rep proteins, Rep 78 and Rep 52, bind and inhibit the cyclic AMP responsive cellular kinases, PrKX and PKA. However, the spliced form of Rep 78, Rep 68, retains the apoptotic inducing activities, although at a reduced level. Thus, it appears that Rep proteins induce apoptotic cell death by accumulated effects due to various activities, e.g. the endonuclease activity of Rep 68 and Rep 78 may not be necessary for apoptosis. By evaluating different mutated forms of the Rep proteins, the biochemical activities required for inducing cell death can be isolated. Biochemical activities of Rep proteins. We have recently characterized features of the Rep proteins that may provide a basis for explaining the aspects of AAV life-cycle that remained obscure. All four of the Rep proteins share the ATP binding pocket. We have shown that the two smaller Rep proteins which lack the specific DNA binding domain contained in the two larger Rep proteins, are able to function as DNA helicases. This is consistent with the putative role of these Rep proteins in packaging the single- stranded DNA genome into the assembled capsids. More recently, we have demonstrated that the p5 derived Rep proteins function as DNA nicking and re-joining enzymes. Thus, these proteins may be defined formally as toposiomerases. All single-stranded DNA genomes replicate through the use of replication initiator proteins that function similarly regardless of the phylogenetic distribution. For example, bacteriophage M13, geminivirus of plants, or parvoviridae, encodes a protein which recognizes the cognate origin of DNA synthesis. The replication protein binds to the origin, nicks one strand permitting a DNA polymerase to initiate leading-strand DNA synthesis. Thus, AAV Rep proteins contain the activities described for the prokaryotic and plant replication proteins. The ability of AAV Rep protein to specifically nick and rejoin DNA is likely to be involved directly in AAV DNA replication as well as targeted integration of AAV into human chromosome 19. - adeno- associated virus, AAV, replication initiator proteins